Poultry vaccine

ABSTRACT

This invention relates to a novel infectious bronchitis virus (IBV) serotype and to attenuated IBV strains derived therefrom, and also to live or inactivated vaccines made using such IB virus.

This is a continuation of application Ser. No. 08/282,689 filed Aug. 1,1994, now abandoned.

The present invention relates to a new infectious bronchitis virusserotype, to attenuated infectious bronchitis virus strains derived fromthis new serotype and to a live vaccine, for use in immunizing poultry,which vaccine contains the attenuated strain of infectious bronchitisvirus. The invention also relates to an inactivated vaccine containingeither the new serotype which has been inactivated, or an inactivatedattenuated strain derived therefrom. The invention is also concernedwith a process for the preparation of live infectious bronchitisvaccines.

Infectious bronchitis virus (IBV) is a member of the genus coronavirusof the family Coronaviridae. The virus is usually about 40-100 nm insize, being round with projecting 20 nm spikes. IBV is the causativeagent of an acute, highly contagious disease in chickens of all ages,affecting the respiratory, reproductive and renal systems.

IBV has been reported in all countries where an intensive poultryindustry has been developed. Young chickens up to 4 weeks of age aremost susceptible to respiratory disease, infection leading to high ratesof morbidity and to mortality resulting from secondary bacterialinfection. Infection in layers results in a drop in egg production, orfailure to lay at full potential, together with an increase in thenumber of down-graded eggs with thin, misshapen, rough and soft-shellsproduced. Although layers usually recover from the disease, their eggproduction rarely returns to pre-infection levels. Thus infection offlocks of chickens with IBV can have a serious economic effect.

Spackman and Cameron (Veterinary Record, (1983), 113,354-355.) isolatedIBV from pheasants with a history of respiratory signs and aberrant eggproduction. This disease problem in pheasants was successfullycontrolled by the use of oil-based inactivated IBV vaccine. Thus theterm poultry, as used herein, is intended to embrace chickens, pheasantsand any other domesticated bird serving as a source of eggs or meat andthat are susceptible to infection by IBV.

Initially, most of the virulent IBV strains isolated were of theMasachussetts serotype, and for many years this was thought to be theonly serotype of IBV. However, it is now known that different serotypesappear to be present in distinct geographical areas; for example,Arkansas 99 (Johnson et al.,Avian Dis.,(1973),17,518-523) has beenisolated in the USA, whilst D274 serotype (Davelaar et al.,Proc. WorldVet. Poultry Assoc.,Oslo,1981,p.44) has not been isolated in the USA.

The only practical means of preventing infectious bronchitis in poultryis to vaccinate against the infection. Two main types of vaccine areavailable and they are attenuated and inactivated.

Reports of new serotypes have been numerous over the years in manycountries. More recently, for example, Gough et al., in The VeterinaryRecord, (1992). 130, 493-494, reported a possible new strain of IBV ininfected domestic fowl in Great Britain. They showed that the newstrain' of IBV had antigenic differences from M41 and the Dutch variantsfrom which many current IBV vaccines are derived.

In addition, Parsons et al., in The Veterinary Record, (1992), 131,408-411 also described isolates of IBV which "were serologicallydistinct from isolates previously described and capable of causingcharacteristic infectious bronchitis-like respiratory infection in youngchickens."

It is well known that many new serotypes will not cause infectiousbronchitis in birds which have been vaccinated. However, a new serotypeof IBV has now been isolated which has been found to cause overtinfection in vaccinated chickens.

Neither of these two recent publications describe the serotype or strainin such a manner as to enable the skilled person in the art to be ableto identify an isolate of IBV as belonging to this new serotype.

According to one aspect of the invention there is provided a novelinfectious bronchitis virus serotype which has been isolated fromchickens and deposited on Jul. 6, 1993 with the European Collection ofAnimal Cell Cultures, Porton Down, United Kingdom, under the BudapestTreaty, and designated accession no. V93070612, and/or an infectiousbronchitis strain which is cross-neutralised significantly by antiseraraised in chickens against said deposited strain.

According to a second aspect of the invention there is provided an IBVstrain belonging to a new serotype, said new serotype beingcharacterised by the deposit referred to above, said IBV strain beingcross-neutralised significantly by antisera raised in chickens againstsaid deposited strain.

The novel IBV serotype of the present invention, hereinafter referred toas 4/91, was isolated from chickens which had previously been vaccinatedwith a commercially available inactivated bivalent infectious bronchitisvaccine, and which exhibited the characteristic symptoms of IBVinfection. Samples of tissue from the trachea and caecal tonsils fromdead affected birds were taken, homogenised and passaged in chickenembryo tracheal organ cultures. After the second or third organ culturepassage, virus particles with typical coronavirus morphology wereobserved by electron microscopy in organ culture supernatants.

In addition to identification using the electron microscope, theisolated virus was found to possess nucleic acid of the RNA type. Usingan ELISA it was demonstrated that the viruses contained antigen incommon with known avian coronavirus, e.g. IBV reference strain M41. Thevirus was also identified as being IBV by performing the polymerasechain reaction with viral RNA using universal primers for IBV accordingto Lin, Z.,Archives of Virology, (1991),116,19.

Serum neutralisation tests were carried out in tracheal organ cultures.Monospecific antiserum to each of 41 known serotypes was prepared inchickens and used to test the novel serotype 4/91. Table 1 summarisesthe results of the virus neutralisation tests. The isolated virus wasalso found to spontaneously hemagglutinate chicken red blood cells andthis hemagglutination could be inhibited by specific antiserum.

Cross neutralisation tests were carried out between the novel IBVserotype 4/91 and seven other IBV serotypes. This was carried out intracheal organ cultures. Each monospecific antiserum being tested at arange of doubling dilutions against log10 2.0 CD50 of each virus(CD50=median ciliostatic dose). The results are presented in Table 2.

Table 3 shows the results of analysing the data according to the methodof Archetti,I. and Horsfall, F. L.,J. Expt. Med.,(1950),92,441-462,which expresses the relationship (r) between strains as a percentage.Applying this method to IB neutralisation tests in embryos, it has beensuggested (Locher et al.,Berliner und Munchener TierarztlicheWochenschrift,(1983),96,269-274) that r values greater than 50 indicateclose relationships, values between 25 and 50 indicate a lower degree ofrelationship, whilst values below 25 indicate little relationship.

Thus the term "cross-neutralised significantly" means that, using themethod of Archetti and Horsfall, an IBV strain with an `r` value ofgreater than 50 may be considered to be closely related to the novelserotype of the present invention.

The present invention also relates to an attenuated infectiousbronchitis virus, that is the IBV serotype 4/91, or an IBV strainbelonging to the new serotype, as defined above, which is attenuated.Such an attenuated IBV is obtainable by passaging the IBV in a cultureon a suitable medium a sufficient number of times to reduce itspathogenicity whilst retaining its immunogenicity. Preferably the IBV ispassaged at least 30 times.

An attenuated strain of the novel IBV serotype has been deposited onJul. 6, 1993 with the European Collection of Animal Cell Cultures,Porton Down, UK,under the Budapest Treaty, under accession no.V93070611.

To attenuate the novel IBV serotype of the present invention the virusmay be passaged in embryonated eggs. Inoculation of the eggs can be viathe allantoic cavity, chorioallantoic membrane, yolk sac, amnioticcavity or even direct into the embryo. The virus can be passaged atregular intervals of from 7 hours up to 4 days. More usually passagingtakes place between 16 to 36 hours, preferably every 24 hours.

Alternatively, attenuation may be achieved by passaging the virus inavian cell culture, such as chick embryo kidney cells.

The attenuated IBV prepared as described above, or the depositedattenuated strain, may be used in the preparation of a live vaccine.Thus according to a further aspect of the present invention there isprovided a live infectious bronchitis vaccine for use in immunizingpoultry said vaccine derived from the IBV described above.

According to another aspect of the invention there is provided a processfor the preparation of a live infectious bronchitis vaccine whichcomprises passaging the novel IBV serotype, or strain as hereinbeforedescribed, in a culture on a suitable medium a sufficient number oftimes to reduce its pathogenicity whilst retaining its immunogenicityand processing the material harvested to produce a vaccine. Preferablythe virus is passaged at least 30 times.

The present invention also relates to the use of an attenuatedinfectious bronchitis virus strain, as hereinbefore described, for thepreparation of a vaccine for use in vaccinating poultry against IBV.

Such live vaccines may be administered by eye drop, nose drop, indrinking water, or by spraying the birds, at any age from one day old upto point of lay (about 18 weeks). The dosage used is preferably in therange of log10 3.0 to log10 7.0 EID50 per bird, preferably between log104.0 and log10 5.0 EID50 per bird.

Such an attenuated IB vaccine may be administered in combination withother live avian vaccines, for example Newcastle Disease Virus (NDV),Mareks Disease Virus (MDV), Infectious Bursal Disease (IBD), reovirus,Avian Encephalomyelitis, Chicken Anaemia Agent (CAA) and other IBVserotypes.

Alternatively, the novel IBV serotype according to the invention and/orthe novel attenuated IBV strain described above may be presented as aninactivated vaccine. According to yet a further aspect of the inventionthere is provided an inactivated infectious bronchitis vaccine for usein immunizing poultry, which vaccine contains IBV which is derived fromthe novel IBV serotype described above, the IBV strain as hereinbeforedefined or the attenuated IBV strain described above.

For both live and inactivated vaccine production the IBV is usuallygrown in embryonated specific pathogen free (SPF) chicken eggs. Afterharvesting, the virus may be inactivated, for use in a killed vaccine,using for example formaldehyde or β-propiolactone.

After inactivation and, if necessary, adjusting of the pH andneutralising of the inactivating agent, the inactivated virus may bemixed with an adjuvant. The adjuvant can be aluminium hydroxide or acomposition consisting of mineral oil (e.g. Marcol 82) or a plant oiland one or more emulsifiers like Tween 80 and Span 80.

Inactivated vaccines are usually administered by subcutaneous orintramuscular injection at between 10 to 20 weeks of age. Inactivatedvaccine may contain the antigenic equivalent of log10 5.0 to log10 8.0EID50 per bird dose, preferably log10 6.0 to log10 8.0 EID50.

Such an inactivated IBV vaccine may be administered in combination withother inactivated avian vaccines, for example NDV, CAA, Egg DropSyndrome 1976 and other IBV serotypes.

According to yet a further aspect of the invention there is provided amethod for protecting poultry against IBV comprising administering avaccine as hereinbefore described to susceptible birds.

The invention is illustrated by the following examples.

EXAMPLE 1.

Isolation of Infectious Bronchitis Virus.

Samples of trachea, kidney and caecal tonsils were taken from dead birdsfrom flocks manifesting the clinical symptoms of infection with IBV. Thetissue samples were homogenised in Eagle's serum free medium, containingpenicillin and streptomycin, to give 10% (w/v) suspension.

After centrifugation at 1,500 g for 15 min., 0.1 ml of supernatant wasinoculated into each of 10 drained tracheal organ cultures (TOC). After1 hr. adsorption at 37° C., 0.5 ml of Eagle's serum free medium wasadded to each culture. The TOC were incubated at 37° C. on a roller drumand examined daily for ciliostasis. Up to three `blind` passages werecarried out before a sample was discarded as negative.

IBV was indicated in samples where ciliostasis of the TOC was observed.Supernatants of `positive` samples were checked for absence ofhaemagglutination (to ensure freedom from Newcastle Disease virus), thentheir identity as IB was confirmed by serological analysis.

An ELISA assay was used to characterise further the isolates (seeMockett. A. P. A. and Cook, J. K. A.,Avian Patholoqy,(1986),15,437.).Antiserum was raised to serotype 4/91 in specific pathogen free RhodeIsland red chickens inoculated intranasally and bled four weeks later.The monospecific antiserum raised to isolate 4/91 was shown to react toa titre of log2 13.0 in the ELISA test, using plates coated with the M41strain of IBV, evidence that the isolates were infectious bronchitisvirus.

EXAMPLE 2.

Attenuation of IBV Strain 4/91.

SPF white leghorn embryonated eggs, used throughout at between 8 and 11days of preincubation, were initially inoculated with 0.1 ml ofpathogenic IBV 4/91 containing log10 6.4 CD50/ml of virus via theallantoic cavity and incubated at 37° C. for 24 hours. The allantoicfluid of half the embryos per group was harvested at the appointed time.The remaining embryonated eggs were incubated until 17-18 days old whenthey are examined for abnormalities indicative of IBV infection.

The harvested allantoic fluid, at appropriate dilution, was inoculated(0.1 ml) into the allantoic cavity of 10 embryos and harvested, asabove, after 24 hours incubation. This passaging was carried outcontinuously until a sufficient number of passages (at least 30) hadbeen achieved, with incubation times varying from 7 to 36 hours.

At selected passages, the allantoic fluid that had been harvested wastitrated. A series of 10-fold dilutions of the fluid was prepared andassayed in either 9-11 day old embryos (inoculated via the allantoicroute) or chicken embryo tracheal organ culture (TOC).

At intervals, the identity of the passaged virus was checked by means ofa neutralisation test in TOC. Serial 2-fold dilutions of a knownpositive 4/91-specific chicken antiserum were mixed with an equal volumeof embryo passaged IBV 4/91 (log10 2.0 CD50), incubated at 37° C. for 1hour then inoculated into drained TOC (5/dilution). After a 1 houradsorption period at 37° C. the TOC were overlaid (0.5 ml) with EaglesMEM containing hepes and α-methyl glucoside, then incubated at 37° C.and read 3 to 4 days later.

EXAMPLE 3.

Preparation and Assessment of Attenuated IBV Vaccine.

The attenuated IBV prepared as described in Example 2 was harvested fromthe allantoic cavity. The allantoic fluid was clarified bycentrifugation and/or filtration and subsequently processed into asuitable vaccine preparation by methods known per se.

Groups of 9-day-old chicks were inoculated by eyedrop (0.1 ml) witheither the attenuated IBV vaccine described above, or the Massachusettsvaccine (H120) or MA5 vaccine. At 30 days of age the chicks werechallenged by inoculation with log10 4.6 CD50 of the IBV serotype 4/91grown in tracheal organ culture. Seven days later 5 chicks/group werekilled and their tracheas removed for the "ciliostasis" test (seeDarbyshire, J. H.,Avian Pathology,(1980),9,179-184.).

The chicks were killed by intravenous injection of Euthatal. Theirtracheas were carefully removed and, using a scalpel, 10 rings carefullycut (3 from the top, 4 from the middle and 3 from the bottom). The 10rings were examined under a low magnification and each scored on a scalefrom 0 (100% ciliary activity) to 4 (100% ciliostasis).

Results.

The results summarised in Table 4 show that at the one time tested (7days post challenge) the attenuated IBV strain prepared in Example 2protected well against a high homologous challenge. The two vaccinestrains (H120 and MA5) gave poor protection against this high challenge;in each case 1/5 chickens was protected.

EXAMPLE 4.

Preparation of Inactivated IB Vaccine.

IBV was inoculated, via the allantoic cavity, into embryonated eggswhich had been pre-incubated for between 8 and 11 days. Afterapproximately 1-3 days incubation, the allantoic fluid was harvested,pooled, clarified by centrifugation and the virus was inactivated by theaddition of formalin. The inactivation of the IB virus was confirmed byinoculation of 9 day old embryonated eggs via the allantoic cavity andsubsequent examination of the embryos for signs of IB infection. An oilemulsion vaccine was then prepared using standard procedures. This wasinoculated either subcutaneously or intramuscularly into groups ofchickens, which may or may not have been `primed` by earlieradministration of live-attenuated IB vaccine. These chickens were bledat least 7 weeks later and their sera examined for IB specificantibodies by an ELISA.

    ______________________________________                                        1)    Unprimed, given inactivated vaccine,                                                               Mean titre (log2) 9.1                                    bled 7 weeks later.  Range log2 7.6-10.6                                2)    Primed, given inactivated vaccine,                                                                 Mean titre (log2) 12.6                                   bled 7 weeks later.  Range log2 11.6-13.6                               ______________________________________                                    

Table 1

IB virus serotype 4/91 was tested in a neutralisation test againstmonospecific antiserum raised against each of the following IBVserotypes.

In each case the neutralisation titre was <1:8.

UK serotypes A; B; C; D; F; F1; H; I; J; 690; 317; 183; 918; 225;HV1-116; D41; VF70-861, Allen

Dutch D207; D3896; D3128; D212

Portugal 135355/82

Israel IBV-11

Germany 5423/86; 332/88; IBV-10

Italy 1767/84; Forli 1; Forli 2

Belgium B-1486

USA Massachusettes; Connecticut; Iowa 609, Iowa 97; Holte; Gray;Arkansas; 632

South Africa 890/80; 145/86

                                      TABLE 2                                     __________________________________________________________________________    Cross neutralisation between IBV serotype 4/91 and other IBV serotypes        Virus (log.sub.10 2.0 CD.sub.50)                                                          UK      France            Morocco                                                                             Australia                         Antiserum                                                                            4/91 591 E   CR84084                                                                             CR84221                                                                             CR88061                                                                             G     T                                 __________________________________________________________________________    4/91   1600*                                                                              --  11  ND    --    30    9     --                                UK 591 13   1000                                                                              --  ND    ND    ND    --    --                                UK-E   46   --  6260                                                                              --    14    --    --    --                                Fr.84084                                                                             --   --  --  720   10    --    --    15                                Fr.84221                                                                             30   ND  70  42    3240  10    --    14                                Fr.88061                                                                             10   ND  --  --    --    140   10    --                                Morocco G                                                                            19   --  --  --    --    --    400   --                                Australia T                                                                          20   ND  16  58    10    ND    --    1300                              __________________________________________________________________________     *Reciprocal antiserum titre (homologous reaction underlined)                  -- titre = <1:8                                                               ND not done                                                              

                                      TABLE 3                                     __________________________________________________________________________    Results analysed by the method of Archetti and Horsfall (1950)                "r" values                                                                               UK      France            Morocco                                                                             Australia                                 4/91                                                                              591 E   CR84084                                                                             CR84221                                                                             CR88061                                                                             G     T                                  __________________________________________________________________________    4/91   100 --  2   ND    --    5     --    --                                 UK 591     100 --  ND    ND    ND    --    ND                                 UK-E           100 --    --    --    --    --                                 Fr.84084           100   --    --    --    3                                  Fr.84221                 100   --    --    --                                 Fr.88061                       100   --    ND                                 Morocco G                            100   --                                 Australia T                                100                                __________________________________________________________________________     -- = ≦1                                                           

                  TABLE 4                                                         ______________________________________                                        Ciliary activity score on tracheas of 5 birds killed 7 days                   after challenge with the virulent strain of IBV 4/91                          original inoculum (3 weeks pre challenge)                                     Attenuated 4/91     H120    MA5                                               ______________________________________                                                 1*              5       6                                                    1               16      18                                                    2               21      20                                                    2               31      22                                                    5               40      40                                            Total   11              113     106                                           Mean      2.2             22.6    21.6                                        ______________________________________                                         *Total score for 10 rings examined from each individual bird.            

I claim:
 1. An isolated infectious bronchitis virus (IBV), which is of the same serotype as that of the IBV strain 4/91 deposited at the European Collection of Animal Cell Cultures, Porton Down, UK, under accession no. V93070612, wherein the IBV strain is cross-neutralized by antisera raised in chickens against said deposited IBV, to the extent that said strain has an r value of greater than 50 by the method of Archetti and Horsfall.
 2. The infectious bronchitis virus according to claim 1, which is deposited at the European Collection of Animal Cell Cultures, Porton Down, UK, under accession no. V93070612.
 3. The infectious bronchitis virus according to claim 1, wherein the virus is attenuated.
 4. The attenuated infectious bronchitis virus according to claim 3, obtained by passaging the infectious bronchitis virus in a culture on a suitable medium a sufficient number of times to reduce its pathogenicity while retaining its immunogenicity.
 5. The attenuated IBV according to claim 4 obtained by passaging the virus at least 30 times.
 6. An attenuated infectious bronchitis virus, which is deposited at European Collection of Animal Cell Cultures, Porton Down, UK under accession no. V93070611. 